Inefficient fast PCR

Details Inefficient fast PCR Inefficient fast PCR

1999-04-06

2001-10-16

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S005000, C435S006120, C435S007100, C435S021000, C435S028000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06303343

ABSTRACT:

CROSS-REFERENCES TO RELATED APPLICATIONS
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STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable
FIELD OF THE INVENTION
This invention is in the field of cyclic polymerase-mediated reactions such as PCR. More specifically, this invention relates to altering the time within which the steps of such reactions are carried out. The methods of this invention are particularly relevant to reactions such as PCR as performed in devices that allow very short cycle times, such as microfluidic devices.
BACKGROUND OF THE INVENTION
PCR and other cyclic polymerase-mediated reactions are standard tools of modern biological research, and are also commonly used for numerous applications including medical diagnostic procedures and forensic applications. PCR is based on three discrete, multiply repeated steps: denaturation of a DNA template, annealing of a primer to the denatured DNA template, and extension of the primer with a polymerase to create a nucleic acid complementary to the template. The conditions under which these steps are performed are well established in the art.
Generally, standard PCR protocols teach the use of a small number of cycles (e.g. 20-35 cycles) which are optimized for maximum efficiency in each cycle, i.e. to ensure that a highest possible percentage of template molecules is copied in each cycle. Typically, this entails cycle times of 1, 2, or more minutes. For example, the standard reference Innis et al.,
PCR Protocols, A Guide to Methods and Applications
(Academic Press, Inc.; 1990)(“Innis”) suggests the following conditions under the heading “Standard PCR Amplification Protocol” (at page 4):
Denaturation
96° C., 15 seconds
Primer Annealing
55° C., 30 seconds
Primer Extension
72° C., 1.5 minutes
Such times, or longer, are typical in the field. Similar protocols can be found in, e.g. Sambrook et al. (1989)
Molecular Cloning, A Laboratory Manual
(2d Edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (“Sambrook”), which teaches a 6 minute cycle, and Ausubel et al., eds. (1996)
Current Protocols in Molecular Biology,
Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (“Ausubel”), which teaches a 5 minute cycle. Accordingly, only up to about 20 or 35 cycles are typically required to generate a detectable and/or isolatable amount of product.
Recently, attempts have been made to shorten the time required for each cycle of a PCR. Such methods often reduce the time by, for example, performing the PCR in devices that allow rapid temperature changes, thereby eliminating much of the time previously required for PCR to “ramp” the temperature of the solution from one stage of the PCR to the next. In addition, it has been recognized that the use of apparatus that allow greater heat transfer, e.g. thin-walled tubes, turbulent air-based machines, also allow the use of shorter cycle times. For example, the RapidCycler™, from Idaho Technologies, Inc. allows relative rapid ramping times between each temperature of a PCR and relatively efficient thermal transfer from the cycler to the samples. Accordingly, the Idaho Technologies Internet site (www.idahotec.com) provides an example of a PCR, wherein 30 cycles were completed in slightly less than 10 minutes.
Another example was discussed by Kopp et al. (1998)
Science,
280:1046. Kopp et al. describe a microfluidic continuous flow PCR system where the PCR reactants were flowed through a chip having three discrete temperature zones. A channel was fixed within the chip to allow a fluid within the channel to pass through each of the zones repeatedly, generating a PCR comprising 20 cycles. By varying the speed by which the fluids flowed through the channel, Kopp et al. created a series of PCRs, each with cycles of varying lengths. Because of the design of this system, the reagents within the channel underwent essentially instantaneous changes in temperature. Thus, the cycle time in this system reflected the time at each temperature, with no substantial temporal contribution from the ramping times. Kopp et al. performed a series of 8 reactions, with cycle times varying from 60 to 4.5 seconds.
Consistent with previous studies, the shorter cycles used by Kopp et al. resulted in a significantly decreased amount of product. For example, a cycle time of about 12 seconds generated only about 45% of the product generated by a PCR using a 56 second cycle. A cycle time of 6.6 seconds generated less than about 10% of the 56-second cycle product. A cycle time of 4.5 seconds did not yield any detectable product.
None of these examples have challenged the teaching, well known to those of skill in the art, that regardless of the duration of the cycle, it is desirable to maximize the efficiency of the cycle. Accordingly, even those applications that suggest a low cycle time invariably suggest a standard, low number of cycles. For example, the system used by Kopp et al. was limited to 20 cycles, regardless of the length of the cycle. Similarly, the RapidCycler specifications page suggests using 30 cycle reactions. According to Kary Mullis, the Nobel Prize winning inventor of PCR (as quoted in Innis, supra), “If you have to go more than 40 cycles to amplify a single-copy gene, there is something seriously wrong with your PCR.”
This invention is based, in part, on the surprising discovery that it is often desirable to perform PCR using short inefficient cycles. Specifically, despite their relative inefficiency, when short, inefficient cycles are repeated an unconventionally high number of times, it is possible to generate more product in the same amount of time or in less time than under standard conditions.
SUMMARY OF THE INVENTION
This invention is based on the surprising discovery that cyclic polymerase-mediated reactions, such as PCR, can be effectively carried out using very short cycles. As described herein, such reactions can be productively carried out even when the cycles are truncated to the point where they are significantly less efficient than under standard conditions. In particular, this invention demonstrates that performing a cyclic polymerase-mediated reaction using a higher than standard number of such short, inefficient cycles yields a high amount of product. In many cases, the amount of product generated using a high number of short cycles is greater than the amount generated in the same overall amount of time using standard conditions.
This invention teaches methods for performing cyclic polymerase-mediated reactions, wherein template molecules, polymerase enzymes, and primer molecules are incubated so as to extend the primer molecules, thereby duplicating at least a fraction of the template molecules. In these methods, the time allowed for the denaturation of the template and/or extension of the primer is less than under standard conditions, which are generally designed for optimum efficiency, i.e. maximum duplication of the template molecules. Consequently, when performed according to the methods described herein, each cycle of such a reaction is significantly less efficient than is typically accomplished using standard techniques in the art. In preferred embodiments, the percentage of template molecules that are duplicated in the short cycles steps is e.g. 90%, 70%, 50%, 30%, 10%, 5%, or less. Such cycles may be as short as 8-10, 6, 5, 4.5, 4, 2, 1, 0.5 seconds or less.
Because of the relative inefficiency of such short extension steps, the steps are repeated more times than is generally taught according to standard PCR protocols. In certain embodiments, the reaction comprises 30, 50, 70, 100, 200, 400, 1000, or more cycles.
In particularly preferred embodiments of this invention, cyclic polymerase-mediated reactions are performed using a high number of short, inefficient extension steps e.g. in a microfluidic device.
In certain embodiments of this invention, these processes are accomplished by changing the temperature of the solution containing the templates, primers, and polymerase. In such embodiment

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